Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

BACKGROUND: The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. METHODS: The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. RESULTS: A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. CONCLUSIONS: The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run.

Correlation Between Virulence Genotype and Fluoroquinolone Resistance in Carbapenem-Resistant Pseudomonas aeruginosa

BACKGROUND: Pseudomonas aeruginosa is a clinically important pathogen that causes opportunistic infections and nosocomial outbreaks. Recently, the type III secretion system (TTSS) has been shown to play an important role in the virulence of P. aeruginosa. ExoU, in particular, has the greatest impact on disease severity. We examined the relationship among the TTSS effector genotype (exoS and exoU), fluoroquinolone resistance, and target site mutations in 66 carbapenem-resistant P. aeruginosa strains. METHODS: Sixty-six carbapenem-resistant P. aeruginosa strains were collected from patients in a university hospital in Daejeon, Korea, from January 2008 to May 2012. Minimum inhibitory concentrations (MICs) of fluoroquinolones (ciprofloxacin and levofloxacin) were determined by using the agar dilution method. We used PCR and sequencing to determine the TTSS effector genotype and quinolone resistance-determining regions (QRDRs) of the respective target genes gyrA, gyrB, parC, and parE. RESULTS: A higher proportion of exoU+ strains were fluoroquinolone-resistant than exoS+ strains (93.2%, 41/44 vs. 45.0%, 9/20; P< or =0.0001). Additionally, exoU+ strains were more likely to carry combined mutations than exoS+ strains (97.6%, 40/41 vs. 70%, 7/10; P=0.021), and MIC increased as the number of active mutations increased. CONCLUSIONS: The recent overuse of fluoroquinolone has led to both increased resistance and enhanced virulence of carbapenem-resistant P. aeruginosa. These data indicate a specific relationship among exoU genotype, fluoroquinolone resistance, and resistance-conferring mutations.

Comparison of antibiotic resistance and virulence factors in pigmented and Non-pigmented Pseudomonas aeruginosa / Comparación de la resistencia antibiótica y los factores de virulencia en las Pseudomonas aeruginosa pigmentadas y no pigmentadas

OBJECTIVES: Pseudomonas aeruginosa produces multiple virulence factors that have been implicated in pathogenesis and quorum sensing. The aim of this study was to determine differences in the virulence factors of pigmented and non-pigmented P aeruginosa isolates. METHODS: Associations were assessed between pigment production (pyocyanin and pyoverdin) and production of DNase, elastase, lipase, protease, siderophore, twitching motility, antibiotic resistance patterns and virulence-associated genes in 57 non-duplicate P aeruginosa isolates from wounds, sputum, urine, high vaginal swab (HVS), ear, eye and respiratory tract swabs and aspirates of peritoneum and ulcers. RESULTS: Most (82.5%) of the isolates produced either pigment. Pigmented isolates produced more frequently and significant more (p < 0.05) DNase, elastase, lipase protease, and siderophore. Imipenem was the only antibiotic to which all isolates were susceptible (p < 0.05), while 93% and 32% were resistant to tetracycline and norfloxacin, respectively. There was however no significant difference between pigmented and non-pigmented isolates when antibiotic resistance was compared. While isolates had multiple virulence-associated genes, exoS (51%), rhlA (37%) and rhlB (46%) were the predominant genes detected. Except for exoY, genes were present in pigmented isolates more frequently than in non-pigmented isolates. CONCLUSION: The results of this study suggest that antibiotic resistance per se might not be associated with the pigment production in P aeruginosa. However, pigment production appeared to be more significantly associated with multi-drug resistance, presence ofvirulence-associated genes, and expression ofcertain virulence factors, most notably elastase, protease, siderophore and DNase activity. Since pigment production is easy to determine, this might to be a good starting point to identify the virulence status ofan isolate.

OBJETIVO: Pseudomonas aeruginosa produce múltiples factores de virulencia que han estado implicados en patogénesis y detección de quórum (quorum sensing). El objetivo de este estudio fue determinar las diferencias en los factores de virulencia de aislados de P aeruginosa pigmentada y no pigmentada. MÉTODO: Se evaluaron las asociaciones entre la producción de pigmentos (piocianina y pioverdina) y la producción de Dnasa, elastasa, lipasa, proteasa, sideróforos, motilidad asociada a superficies (twitching), patrones de resistencia antibiótica, y genes asociados con virulencia en 57 aislados de P aeruginosa no duplicados, de heridas, esputo, orina, exudado vaginal, exudados de oídos, ojos, y vías respiratorias, y aspirados de peritoneo y úlceras. RESULTADOS: La mayor parte (82.5%) de los aislados produjeron uno de los pigmentos. Los aislados pigmentados produjeron con mayor frecuencia y más significativamente (p < 0.05). Dnasa, elastasa, lipasa, proteasa, y siderósforos. Imipenem fue el único antibiótico al que todos los aislados eran susceptibles (p < 0.05), mientras que el 93% y el 32% fueron resistentes a la tetraciclina y a la norfloxacina, respectivamente. Sin embargo, no hubo diferencia significativa entre los aislados pigmentados y los no pigmentados cuando se comparaba la resistencia antibiótica. Si bien los aislados tenían múltiples genes asociados con la virulencia, exoS (51%), rhlA (37%) y rhlB (46%) fueron los genes predominantes detectados. Con excepción de exoY, los genes estuvieron presentes en aislados pigmentados con mayor frecuencia que en los aislados no pigmentados. CONCLUSIÓN: Los resultados de este estudio sugieren que la resistencia antibiótica per se podría no estar asociada con la producción de pigmentos en P aeruginosa. Sin embargo, la producción de pigmentos parecía estar asociada más significativamente con la resistencia a las multidrogas, la presencia de genes asociados con la virulencia, y la expresión de ciertos factores de virulencia, en particular la actividad de la elastasa, la proteasa, los sideróforos, y la Dnasa. Puesto que la producción de pigmentos es fácil de determinar, esto podría ser un buen punto de partida para identificar el estado de virulencia de un aislado.

The mucosal adjuvanticity of two nontoxic mutants of Escherichia coli heat-labile enterotoxin varies with immunization routes

Escherichia coli heat-labile enterotoxin (LT), which causes a characteristic diarrhea in humans and animals, is a strong mucosal immunogen and has powerful mucosal adjuvant activity towards coadministered unrelated antigens. Here we report the different mucosal adjuvanticity of nontoxic LT derivatives, LTS63Y and LTdelta110/112, generated by immunizing through two different mucosal routes. Intragastric (IG) immunization with Helicobacter pylori urease alone resulted in poor systemic IgG and IgA responses and no detectable local secretory IgA, but IG co-immunization with urease and LTdelta110/112 induced high titers of urease-specific local secretory IgA and systemic IgG and IgA, comparable to those induced by wild-type LT. LTS63Y showed far lower adjuvant activity towards urease than LTdelta110/112 in IG immunization, but was more active than LTdelta110/112 in inducing immune responses to urease by intranasal (IN) immunization. LTdelta110/112 predominantly enhanced the induction of urease-specific IgG1 levels following IG immunization, whereas LTS63Y induced high levels of IgG1, IgG2a and IgG2b following IN immunization. In addition, quantitative H. pylori culture of stomach tissue following challenge with H. pylori demonstrated a 90-95% reduction (p < 0.0002) in bacterial burden in mice immunized intranasally with urease using either mutant LT as an adjuvant. These results indicate that the mechanism(s) underlying the adjuvant activities of mutant LTs towards coadmnistered H. pylori urease may differ between the IN and IG mucosal immunization routes.